Disorders of iron metabolism are among the most prevalent cause of human morbidity and mortality worldwide. The recent identification of the gene responsible for hereditary hemochromatosis (HLA-H) represents an important step towards understanding and treating these disorders. The mechanism of action of HLA-H is entirely unknown; it must regulate intestinal iron uptake, but the process is poorly understood. To approach this problem from a novel direction, we took advantage of mice carrying the microcytic anemia mutation (gene symbol mk). These animals are known to have defects in both intestinal and red cell iron uptake. Careful physiology studies performed in a variety of laboratories have indicated that the protein encoded by the mk gene is an important component of the intestinal iron transport apparatus. We mapped the precise chromosomal location of mk using two large backcross panels totally 1000 informative meioses and identified a single candidate gene, Nramp2, that has features of a ATP- dependent transmembrane heavy metal transporter, and is recombinationally inseparable from the phenotype. We have shown that mk animals derived from two distinct spontaneous mutation events both have a glycine to arginine missense mutations in Nramp2 that disrupts a critical transmembrane domain. Taken in the context of the known defects of the mk mouse, our data strongly support the conclusion that Nramp2 is the intestinal iron transporter. It is ubiquitously expressed, and it also appears to be important for bone marrow iron utilization. The work proposed in this application addresses its mechanism of action in iron transport. The specific aims are (1) to characterize the role of Nramp2 in iron metabolism, by investigating its cellular localization, developing an assay for function, carrying out a structure-function analysis and identifying interacting proteins; (2) to determine the effect of the mk mutation on Nramp stability, localization, and function and (3) to rescue the Nramp 2 defect in mk cells and mice by introducing transgenes encoding Nramp2, or its homolog Nramp1, under the control of selected tissue- specific promoters, and (4) to determine whether human patients with similar phenotypic features have mutations in the Nramp2 gene. We anticipate that the results of this characterization will lead to the development of therapeutic agents to modulate intestinal iron absorption, adding a new treatment modality for iron deficiency and iron overload disorders.